AtRKD5 inhibits the parthenogenic potential mediated by AtBBM.

Parthenogenesis, the development of unfertilized egg cells into embryos, is a key component of apomixis. AtBBM (BABY BOOM), a crucial regulator of embryogenesis in Arabidopsis, possesses the capacity to shift nutritional growth toward reproductive growth. However, the mechanisms underlying AtBBM-induced parthenogenesis remain largely unexplored in dicot plants. Our findings revealed that in order to uphold the order of sexual reproduction, the embryo-specific promoter activity of AtBBM as well as repressors that inhibit its expression in egg cells combine to limiting its ability to induce parthenogenesis. Notably, AtRKD5, a RWP-RK domain-containing (RKD) transcription factor, binds to the 3' end of AtBBM and is identified as one of the inhibitory factors for AtBBM expression in the egg cell. In the atrkd5 mutant, we successfully achieved enhanced ectopic expression of AtBBM in egg cells, resulting in the generation of haploid offspring via parthenogenesis at a rate of 0.28%. Furthermore, by introducing chimeric Arabidopsis and rice BBM genes into the egg cell, we achieved a significant 4.6-fold enhancement in haploid induction through the atdmp8/9 mutant. These findings lay a strong foundation for further exploration of the BBM-mediated parthenogenesis mechanism and the improvement of haploid breeding efficiency mediated by the dmp8/9 mutant.

Candidate gene analysis of watermelon stripe pattern locus ClSP ongoing recombination suppression.

KEY MESSAGE: Using two segregating population, watermelon stripe pattern underlying gene ClSP was delimited to a 611.78 Kb region, consisting of four discrete haploblocks and ongoing recombination suppression. Stripe pattern is an important commodity trait in watermelon, displaying diverse types. In this study, two segregating populations were generated for genetic mapping the single dominant locus ClSP, which was finally delimited to a 611.78 Kb interval with suppression of recombination. According to polymorphism sites detected among genotypes, four discrete haploblocks were characterized in this target region. Based on reference genomes, 81 predicted genes were annotated in the ClSP interval, including seven transcription factors namely as candidate No1-No7. Meanwhile, the ortholog gene of cucumber ist responsible for the irregular stripes was considered as candidate No8. Strikingly, gene structures of No1-No5 completely varied from their reference descriptions and subsequently re-annotated. For instance, the original adjacent distribution candidates No2 and No3 were re-annotated as No2_3, while No4 and No5 were integrated as No4_5. Sequence analysis demonstrated the third polymorphism in CDS of re-annotated No4_5 resulting in truncated proteins in non-stripe plants. Furthermore, only No4_5 was down-regulated in light green stripes relative to dark green stripes. Transcriptome analysis identified 356 DEGs between dark green striped and light green striped peels, with genes involved in photosynthesis and chloroplast development down-regulated in light green stripes but calcium ion binding related genes up-regulated. Additionally, 38 DEGs were annotated as transcription factors, with the majority up-regulated in light green stripes, such as ERFs and WRKYs. This study not only contributes to a better understanding of the molecular mechanisms underlying watermelon stripe development, but also provides new insights into the genomic structure of ClSP locus and valuable candidates.

Genome-Wide Identification of CCD Gene Family in Six Cucurbitaceae Species and Its Expression Profiles in Melon.

The carotenoid cleavage dioxygenase (CCD) gene family in plants comprises two subfamilies: CCD and 9-cis-epoxycarotenoid dioxygenase (NCED). Genes in the NCED subfamily are mainly involved in plant responses to abiotic stresses such as salt, low temperature, and drought. Members of the NCED subfamily are the most important rate-limiting enzymes in the biosynthesis of abscisic acid (ABA). In the present study, genome-wide analysis was performed to identify CCD gene members in six Cucurbitaceae species, including watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C.sativus), pumpkin (Cucurbita moschata), bottle gourd (Lagenaria siceraria), and wax gourd (Benincasa hispida). A total of 10, 9, 9, 13, 8, 8 CCD genes were identified in the six species, respectively, and these genes were unevenly distributed in different chromosomes. Phylogenetic analysis showed that CCD genes of the six species clustered into two subfamilies: CCD and NCED, with five and three independent clades, respectively. The number of exons ranged from 1 to 15, and the number of motifs were set to 15 at most. The cis-acting elements analysis showed that a lot of the cis-acting elements were implicated in stress and hormone response. Melon seedlings were treated with salt, low temperature, drought, and ABA, and then tissue-specific analysis of CCDs expression were performed on the root, stem, upper leaf, middle leaf, female flower, male flower, and tendril of melon. The results showed that genes in CCD family exhibited various expression patterns. Different CCD genes of melon showed different degrees of response to abiotic stress. This study presents a comprehensive analysis of CCD gene family in six species of Cucurbitaceae, providing a strong foundation for future studies on specific genes in this family.

Systematic genome-wide analysis of the ethylene-responsive ACS gene family: Contributions to sex form differentiation and development in melon and watermelon.

Ethylene is an important regulatory phytohormone for sex differentiation and flower development. As the rate-limiting enzyme encoding genes in ethylene biosynthesis, ACS gene family has been well studied in cucumber; however, little is known in other cucurbit crops, such as melon and watermelon, which show diverse sex types in the field. Here, we identified and characterized eight ACS genes each in the genomes of melon and watermelon. According to the conserved serine residues at C-terminal, all the ACS genes could be characterized into three groups, which were supported by the exon-intron organizations and conserved motif distributions. ACS genes displayed diverse tissue-specific expression patterns among four melon and three watermelon sex types. Furthermore, a comparative expression analysis in the shoot apex identified orthologous pairs with potential functions in sex determination, e.g., ACS1s and ACS6s. All ACS orthologs in melon and watermelon exhibited similar expression patterns in monoecious and gynoecious genotypes, except for ACS11s and ACS12s. As expected, the majority of ACS genes were responsive to exogenous ethephon; however, some orthologs exhibited opposite expression patterns, such as ACS1s, ACS9s, and ACS10s. Collectively, our findings provide valuable ACS candidates related to flower development in various sex types of melon and watermelon.

The impaired biosynthetic networks in defective tapetum lead to male sterility in watermelon.

Heterosis has been widely applied in watermelon breeding, because of the higher resistance and yield of hybrid. As the basis of heterosis utilization, genic male sterility (GMS) is an important tool for facilitating hybrid seed production, while the detailed mechanism in watermelon is still largely unknown. Here, we report a spontaneous mutant Se18 exhibited complete male sterility due to the uniquely multilayered tapetum and the un-meiotic pollen mother cells during pollen development. Using TMT based quantitative proteomic analyses, a total of 348 differentially abundant proteins (DAPs) were detected with the overwhelming majority down-regulated in mutant Se18. By analyzing the putative orthologs/homologs of Arabidopsis GMS related genes, the biosynthesis and transport of sporopollenin and tryphine precursors were predictably altered in mutant compared to its sibling wild type. Moreover, the general phenylpropanoid pathway as well as its related metabolisms was also expectably impaired in mutant, coincident with the pale yellow petals. Notably, some key transcriptional factors regulating tapetum development, together with their down-regulated targets, offered potentially valuable candidates regarding of male sterility. Collectively, the disrupted regulatory networks underlying male sterility of watermelon was proposed, which provide novel insights into genetic mechanism of male reproductive process and rich gene resources for future research. SIGNIFICANCE: Watermelon is an importantly economical cucurbit crop worldwide, with high nutritional value. Although several male sterile mutants have been identified in watermelon, the underlying molecular mechanism is poorly elucidated. Comparative cytological analysis revealed that the defective development of tapetum was responsible for male sterility in mutant Se18. Combined with the morphological comparison, male floral buds at 2.0-2.5 mm in diameter were confirmed with no obvious phenotypic differences but distinct cytological defects, which were in turn sampled for TMT based proteomic analyses. Referring to functionally characterized GMS related genes, the genetic pathway DYT1-TDF1-AMS-MS188-MS1 regulating tapetum development, together with some downstream targets, were considerably altered in mutant Se18. Moreover, enrichment analyses illustrated the general phenylpropanoid related metabolisms, as well as the biosynthesis and transport of sporopollenin and tryphine precursors, were significantly disrupted in defective anther development. Collectively, the proposed regulatory networks in watermelon not only contribute to a better understanding of molecular mechanisms underlying male sterility, but also provide valuable GMS related candidates for future researches.